JP2010526548A - Signaling peptide - Google Patents

Abstract

  The present invention provides novel peptides of specific sequences and their use as signal peptides or membrane anchored peptides. The invention also relates to chimeric polypeptides comprising one or more such peptides and a polypeptide of interest, as well as nucleic acid molecules, vectors, infectious viral particles and host cells encoding such peptides and chimeric polypeptides. The invention also relates to a pharmaceutical composition comprising such an element and a pharmaceutically acceptable vehicle. The invention also provides a method for recombinantly producing a polypeptide using such a peptide, particularly for expressing the polypeptide of interest immobilized extracellularly or on the surface of the plasma membrane. .

Description

Detailed Description of the Invention

  The present invention relates to novel peptides for use as signal peptides and membrane-anchoring peptides. These peptides are derived from rabies glycoproteins of various virus strains. The present invention also provides nucleic acid molecules and vectors encoding such peptides, as well as such polypeptides of interest that are intended to be secreted from the host cell or fixed to the cell membrane. Also provided are methods of using nucleic acid molecules and vectors. The invention particularly relates to the field of protein expression in various prokaryotic as well as eukaryotic in vitro systems, or animal or human organisms for therapeutic or prophylactic purposes.

  The production of recombinant polypeptides has been one of the major challenges of the biotechnology industry for the last 20 years. Several plasmid DNA and viral vectors are generated to express nucleotide sequences in cultured host cells or living organisms for a variety of purposes, including vaccination, gene therapy, immunotherapy and production of recombinant polypeptides in cultured cells And have been used.

  The purification process is facilitated when the recombinant polypeptide can be recovered from the culture supernatant rather than from the complex protein mixture obtained when cells are disrupted to release intracellular proteins. Secretion of the polypeptide from the transformed host cell is generally advantageous. Furthermore, secretion also reduces the detrimental (eg, toxic) effects that intracellular expression can be retained on the host cell or host organism. Finally, transport through the secretory pathway is also required to undergo post-translational modifications and proteolytic cleavage that are responsible for the stability, folding, immune recognition and biological activity of specific polypeptides.

  As a general guide, secreted polypeptides are expressed as precursors containing a signal peptide at their N-terminus. A signal peptide usually consists of three distinct regions: an N-terminal and positively charged region, a central hydrophobic core region, and a C-terminal cleavage site recognized by signal peptidase. The signal peptide ensures efficient transport of the polypeptide precursor across the endoplasmic reticulum (ER) membrane. Thereafter, the signal peptide is cleaved by signal peptidase during transport to become a mature polypeptide (polypeptide not containing a signal peptide). Once in the ER, the polypeptide is transported to the Golgi apparatus and then follows one of several routes of the secretory pathway, depending on the nature of the polypeptide and the presence of a signaling peptide. For example, the polypeptide can be secreted into an external medium, can be routed to cellular compartments such as endosome and lysosomal compartments, or can be anchored to the cell membrane. It is well known in the art that several post-translational modifications such as glycosylation, disulfide bond formation and proteolytic cleavage of polypeptides occur in the ER and Golgi apparatus. Inappropriate post-translational modifications generally result in inactive polypeptides and inadequate signal peptidase cleavage in the production of heterologous polypeptide products and cannot be used particularly for human use.

  On the other hand, it has been shown previously that the immunogenicity of immunogenic polypeptides can be enhanced by being presented in a form that is anchored to various subcellular compartments or cell membranes (Andrew et al. 1990, J. Virol. 64, 4776-4783), possibly due to increased presentation to the host immune system, mediated by MHC class I and / or MHC class II. For example, Ji et al. (1999, Human Gene Ther. 10: 2727-2740) showed that targeting papillomavirus (HPV) -16 to the endosome / lysosome compartment enhanced its antitumor activity. Report increased production of tumor cell-specific CD4 + helper T cells and CD8 + cytotoxic T lymphocytes in expression host organisms. WO 99/03885 targets HPV-16 E6 and E7 polypeptides at the plasma membrane, increasing their immunogenic performance and thus their therapeutic efficacy in the host organism. Transport and immobilization of membrane bound proteins on the cell surface involve signal peptides and membrane anchored peptides.

  Although several such signaling peptides are available in the art, those obtained from rabies glycoprotein have been found to be effective, particularly with respect to expression mediated by vaccinia virus. Thus, in a variety of host cells and organisms, providing additional signaling sequences that ensure efficient transport through the cell secretion pathway, and / or signal peptidase cleavage, and / or transport and fixation to the cell membrane. It is advantageous.

  Furthermore, the nucleotide sequences encoding the rabies glycoprotein-derived signaling peptides that are the subject of the present invention have been engineered to reduce the percentage of homology between each other to less than 75%, and thus in multigene expression vectors The expressed protein can be secreted from the host cell or immobilized on a cell membrane, such as the plasma membrane, to be present on the cell surface. However, due to the very limited number of effective signaling peptides, particularly signal and membrane anchored peptides, the number of nucleotide sequences that can be expressed from a single vector is limited. In fact, as known to those skilled in the art, recombination events occur between such homologous sequences when the same signaling peptide is used to direct the transport and membrane anchoring of each polypeptide encoded by the multigene expression vector. May result in deletion of nucleotide sequences contained between them (Matzke, M., Matzke, AJM, Scheid, OM In Homologous Recombination and Gene Silencing in Plants; Paszkowski, J. Ed. Kluwer Academic Publishers, Netherlands, 1994; pp 271-300). This instability problem can render vector stocks unusable especially for human clinical trials.

  As detailed above, there is a need in the art for selective signaling peptides for the recombinant production of secreted and membrane-anchored polypeptides, particularly in eukaryotic host cells and organisms. The present invention addresses this need by providing novel peptides derived from glycoproteins of various rabies virus strains as signal and membrane-anchored peptides. Such a peptide has been found to be extremely effective in ordering the secretion and membrane anchoring of a polypeptide of interest expressed in a eukaryotic cell from a vaccinia virus vector. Furthermore, the present invention also provides nucleic acid molecules that encode such peptides whose sequences have been engineered to exhibit a percentage of homology between each other of less than 75%. Use of such engineered nucleic acid molecules to provide secretion and membrane anchoring of multiple polypeptides of interest expressed from a single vector can be achieved naturally when homologous sequences are present in such vector constructs. Reduce the likelihood of homologous recombination events occurring in Thus, the present invention represents an important tool in recombinant technology, particularly with respect to multigene expression vectors, particularly for multi-gene expression vectors, for the secretion and membrane localization of various polypeptides in eukaryotic host cells.

  This technical problem is solved by the provision of the embodiments defined in the claims.

  Other and further aspects, features and advantages of the present invention will become apparent from the following description of the presently preferred embodiments of the invention. These embodiments are presented for purposes of disclosure.

  Thus, in a first aspect, the present invention is from the group consisting of peptides consisting essentially of or consisting of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. Providing a selected peptide.

Throughout this application, “a” and “an” are “at least one”, “at least first”, “one or more”, or “plurality” of the indicated compounds or steps, unless otherwise specified. Is used to mean
As used herein, “and / or” includes the meanings of “and”, “or” and “all of the elements connected by this term, or any other combination”.

  As used herein, “about” or “approximately” means within 20%, preferably within 10%, more preferably within 5% of the indicated value or range.

  As used herein, "comprising" as used for the defined products, compositions and methods, includes those ingredients, compositions and methods including the indicated component or step, It means not to exclude others. “Consisting essentially of” means excluding other components or steps of essential meaning. Thus, a composition consisting essentially of the indicated ingredients does not exclude traces of contaminants and pharmaceutically acceptable carriers. By “consisting of” is meant excluding other components or processes that exceed trace elements. For example, a peptide “consists of” its amino acid sequence if it does not contain any amino acids other than the amino acid sequence shown. A peptide "consists essentially" of its amino acid sequence when the amino acid sequence is present with only a few additional amino acid residues, generally about 1 to about 10 and the like. A peptide "comprises" an amino acid sequence when it is at least part of the last amino acid sequence of the peptide. Such peptides can have from a few to a few hundred additional amino acid residues. Such additional amino acid residues can inter alia play a role in peptide transport, aid in polypeptide production and purification, or extend half-life. The same is true for nucleotide sequences.

  As used herein, the term “amino acid” refers to natural L-amino acids, that is, 20 types of gene-encoded amino acids, alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine ( Ile or I), proline (Pro or P), methionine (Met or M), phenylalanine (Phe or F), tryptophan (Trp or W), glycine (Gly or G), serine (Ser or S), threonine (Thr) Or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), glutamine (Gln or Q), aspartic acid (Asp or D), glutamic acid (Glu or E), lysine (Lys) Or K), arginine (Arg or R And histidine (His or H) and natural derivatives thereof such as β-alanine, ornithine or methionine sulfoxide, or one or more α-amino, α-carboxyl or side chains, such as methyl, formyl, acetyl, glycosyl and phosphoryl Refers to an amino acid modified by the addition of

  As used herein, “polypeptide”, “peptide” and “protein” are used interchangeably and refer to a polymer of amino acid residues comprising nine or more amino acids joined via peptide bonds. The polymer may be linear, branched or cyclic. As a general indicator, if the amino acid polymer is long (eg, more than 80 amino acid residues), it is preferably referred to as a polypeptide or protein. Consequently, a “peptide” is a length of about 9 to about 80 amino acids, advantageously about 10 to about 75 amino acids, preferably about 20 to about 70 amino acids, more preferably about 23 to about 66 amino acids. Refers to a fragment of Peptides can include selected regions of natural proteins, such as those involved in polypeptide localization (eg, directing the polypeptide to a particular cellular compartment or membrane). The definition of “polypeptide / peptide” includes naturally occurring as well as modified polypeptides / peptides.

  As used herein, “natural” refers to material obtained from a natural source (eg, isolated, purified) and is different from material that has been artificially modified or altered by humans in the laboratory. For example, a native polypeptide / peptide is encoded by a gene that is present in the genome of a wild type organism or cell. In contrast, a modified polypeptide / peptide has been modified in the laboratory to differ from the native polypeptide / peptide by, for example, the insertion, deletion or substitution of one or more amino acids, or any combination of these possibilities. Are encoded by different nucleic acid molecules. Where some modifications are intended, they may relate to consecutive residues and / or relate to non-consecutive residues.

  A “signaling peptide” is a peptide that allows for the specific localization of a polypeptide comprising that peptide in a host cell or organism. Examples of signal transduction peptides include signal peptides and membrane-anchored peptides.

  As used herein, “signal peptide” or “signal sequence” refers to an amino acid sequence capable of eliciting a pathway of a polypeptide operably linked to a host cell vesicle (ER). Signal peptides are generally cleaved by endopeptidases (eg, specific ER localization signal peptidases) to release (mature) polypeptides. The length of the signal peptide is generally about 10 to about 40 amino acids, advantageously about 15 to about 30 amino acids, preferably about 18 to about 25 amino acids, particularly preferably about 23 amino acids containing a Met initiator residue. It is a piece.

  As used herein, “membrane-immobilized peptide” refers to a hydrophobic amino acid sequence that can immobilize a polypeptide operably linked to a cell membrane, particularly the plasma membrane. The length of the membrane-anchoring peptide generally ranges from about 20 to about 85 amino acids, advantageously from about 25 to about 75 amino acids, preferably from about 30 to about 70 amino acids, particularly preferably from about 66 amino acids. Preferably, it comprises a highly hydrophobic central domain that can penetrate the lipid bilayer of the cell membrane one or more times or interact with the outer surface of the membrane.

  As used herein, a “chimeric” polypeptide refers to a single polypeptide chain comprising a polypeptide / peptide at a position different from that originally found in the sequence. The various polypeptides / peptides that make up a chimeric polypeptide are usually present in separate proteins and are linked in the chimeric polypeptide, or they may be present in the same protein, but what is a chimeric polypeptide? Exists in different arrangements. For example, a chimeric polypeptide is composed of a signal peptide and a polypeptide of interest obtained from different sources. Another chimeric polypeptide may be composed of a signal peptide, a target polypeptide and a membrane-anchored peptide, the target polypeptide being obtained from a different source than the signal peptide and / or the membrane-anchored peptide.

  “Operatively linked” means that at least two components are juxtaposed, and the components so described are in a relationship to make them function in a predicted manner. For example, a promoter is operably linked to its nucleotide when it induces and allows transcription that allows expression in a host cell or organism. A signal peptide is operably linked to a polypeptide when it induces and allows its transport through the ER and is subsequently cleaved to release the polypeptide. A membrane-anchored peptide is operably linked to a polypeptide when it allows the polypeptide to be immobilized on a cell membrane, particularly the plasma membrane.

  As used herein, a “vector” is an agent capable of delivering and expressing a nucleic acid molecule in a host cell or organism. The vector can be extrachromosomal (eg, episome), integrative (for integration into the host chromosome), autonomously replicating, multiple copies, low copy Be double-stranded, single-stranded, naked or complex with other molecules (eg complexed with lipids or polymers to form liposomes, lipoplexes or It may be a vector having a granular structure such as a nanoparticle, a vector packaged in a virus capsid, a vector immobilized on a solid phase particle, or the like.

  “Nucleic acid”, “nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” are used interchangeably in the present invention, regardless of length, whether polydeoxyribonucleotide (DNA) or polyribonucleotide (RNA) molecule. A polymer of or a combination thereof is defined. This definition includes single or double stranded, linear or circular, natural or synthetic polynucleotides. Furthermore, such polynucleotides are non-natural nucleotides (eg, methylated nucleotides and nucleotide analogs such as those described in US Pat. Nos. 5,525,711, 4,711,955 or EPA 302175). As well as chemical modifications to increase the in vivo stability of the nucleic acid, to enhance its delivery, or to reduce the rate of clearance from the host subject (see, eg, WO 92/03568, US Pat. No. 5,118,672). May be included. If present, modification may be performed before or after polymerization.

  In the present invention, “sequence homology” (or sequence identity) is generally expressed as a percentage and represents nucleotide sequences that retain a certain identity with each other. The percent homology between two nucleotide sequences is a function of the number of identical positions common to those sequences and takes into account the number of gaps that need to be introduced and the length of each gap for optimal alignment. ing. In the art, publicly available in the GCG Wisconsin package and National Center for Biotechnology Information (NCBI) to determine the percentage homology between nucleotide sequences, printed publications (eg, Altschul et al, 1990, Various computer programs and mathematical algorithms such as the Basic Local Aliment Search Tool (BLAST) program described in J. Mol. Biol. 215, 403-410) are available.

  As used herein, “host cell” defines any cell that can be or has become a recipient of the vector of the invention and the progeny of such a cell. The term should be understood broadly to encompass isolated cells, groups of cells, as well as specific constituents of cells, such as tissues or organs. Such cells can be primary, transformed or cultured.

  As used herein, “organism” refers to primates including vertebrates, particularly members of mammalian species, particularly domestic animals, farm animals, sport animals, and humans.

または配列番号２

で示されるアミノ酸配列から本質的になる、またはそれからなる、上記で定義されたシグナルペプチドの機能特性を示す新規な単離されたペプチドを提供する。 In one embodiment, the invention provides SEQ ID NO: 1

Or SEQ ID NO: 2

A novel isolated peptide is provided which exhibits the functional properties of a signal peptide as defined above consisting essentially of or consisting of the amino acid sequence represented by

のアミノ酸配列からなり、配列番号１のシグナルペプチドとは、それぞれ、天然ペプチドではＡｌａ残基が存在するのに対して本発明のシグナルペプチドではＶａＬ残基である５番の位置と、天然ペプチドではＶａｌ残基が存在するのに対して本発明のシグナルペプチドではグリシンである１３番の位置の２箇所で異なる。 More specifically, the signal peptide represented by SEQ ID NO: 1 is derived from the signal peptide present at the N-terminus of the rabies virus ERA glycoprotein precursor disclosed in the UniProt database as accession number M38452. For general information, the natural ERA strain signal peptide is SEQ ID NO: 6.

In the signal peptide of the present invention, the signal peptide of SEQ ID NO: 1 has an Ala residue, whereas the signal peptide of the present invention has a position No. 5 which is a VaL residue. In contrast to the presence of a Val residue, the signal peptide of the present invention differs at two positions of position 13 which is glycine.

のアミノ酸配列からなり、配列番号２のシグナルペプチドとは、天然ペプチドではＬｅｕ残基が存在するのに対して本発明のシグナルペプチドではＧｌｎ残基である４番の位置で異なる。 The signal peptide shown in SEQ ID NO: 2 is derived from the signal peptide present at the N-terminus of the rabies virus PG strain glycoprotein precursor disclosed in the UniProt database as accession number ay009097. For general information, the native PG strain signal peptide is SEQ ID NO: 7.

The signal peptide of SEQ ID NO: 2 differs from the signal peptide of SEQ ID NO: 2 at position 4 which is a Gln residue in the signal peptide of the present invention, while the Leu residue is present in the natural peptide.

  The peptide of the present invention can be modified if necessary as long as the modification does not change the activity of the signal peptide. For example, the initiator Met residue (if the expressed polypeptide contains it) may be deleted.

  The present invention also provides the use of a peptide consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as a signal peptide. The peptides of the present invention are independently used in combination with additional signaling peptides, particularly membrane anchored peptides as described herein, to induce proper transport and immobilization of the polypeptide of interest to the cell membrane. Or make it possible.

で示されるアミノ酸配列から本質的になる、またはそれからなる、上記で定義された膜固定ペプチドの機能特性を示す新規な単離されたペプチドを提供する。 The present invention also provides SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5:

A novel isolated peptide is provided which exhibits the functional properties of a membrane-anchored peptide as defined above consisting essentially of or consisting of the amino acid sequence represented by

のアミノ酸配列からなり、配列番号３の膜固定ペプチドとは３箇所で異なる。 More specifically, the membrane-immobilized peptide represented by SEQ ID NO: 3 is derived from a membrane-immobilized peptide present at the C-terminus of the rabies glycoprotein of the PG strain disclosed in the UniProt database as accession number ay009097. As a summary information, this natural PG strain membrane-fixed peptide is represented by SEQ ID NO: 8

And differs from the membrane-anchored peptide of SEQ ID NO: 3 at three positions.

のアミノ酸配列からなり、配列番号４の膜固定ペプチドとは３箇所で異なる。 The membrane-immobilized peptide represented by SEQ ID NO: 4 is derived from a membrane-immobilized peptide present at the C-terminus of the rabies glycoprotein of the Nishigahara strain disclosed in the UniProt database as accession number S72465. As a summary information, this native Nishigahara strain membrane-fixed peptide is SEQ ID NO: 9

And differs from the membrane-anchored peptide of SEQ ID NO: 4 at three positions.

のアミノ酸配列からなり、配列番号５の膜固定ペプチドとは、天然ペプチドではＴｙｒ残基が存在するのに対して本発明の膜固定ペプチドではＨｉｓ残基である５８番の位置で異なる。 The membrane-immobilized peptide represented by SEQ ID NO: 5 is derived from the membrane-immobilized peptide present at the C-terminus of the rabies glycoprotein of the artic fox strain disclosed in the UniProt database as accession number u11751. As a summary information, this native artic fox strain membrane-fixing peptide is SEQ ID NO: 10.

The natural peptide has a Tyr residue, whereas the membrane-fixed peptide of the present invention differs from the membrane-fixed peptide of SEQ ID NO: 5 at position 58, which is a His residue.

  If necessary, the peptide of the present invention can be modified as long as the activity of the membrane-fixing peptide is not changed.

  The present invention also provides the use of a peptide consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 as a membrane-anchoring peptide. The peptides of the present invention are independently used in combination with one or more additional signaling peptides, particularly signal peptides as described herein, to allow the polypeptide of interest to be immobilized on the cell membrane. Preferably, the peptides of the present invention allow the polypeptide of interest to be immobilized on the plasma membrane and interact with other molecules present on other cell surfaces or extracellular media.

  The present invention also provides a polypeptide of the present invention and (i) a peptide of the present invention consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or (ii) SEQ ID NO: 3, SEQ ID NO: 4 or any of the peptides of the invention consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 5, or (iii) consisting essentially of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or Also provided is a chimeric polypeptide comprising both the peptide of the present invention consisting thereof and the peptide of the present invention consisting essentially of or consisting of the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. .

  Chimeric polypeptides can be made by chemical synthesis or by genetic means, i.e., in-frame fusion of nucleotide sequences encoding each of the various polypeptides / peptides that make up the chimeric polypeptide. “Fusion in frame” means that expression of the fused nucleotide sequence results in a single polypeptide chain that does not include a transcription terminator in between. The fusion may be direct (ie, the codons encoding each polypeptide / peptide are consecutive with no external codons in between) or via a linker ( In the two polypeptide / peptide junctions that make up this chimeric polypeptide). The presence of this linker can aid in the proper formation, folding and / or function of the chimeric polypeptide, or can facilitate the cloning process by introducing one or more suitable restriction sites. As a general guide, linkers are typically 2 to 30 amino acids long and consist of amino acid residues such as glycine, serine, threonine, asparagine, alanine and / or proline. Representative examples of suitable peptide linkers include “Ser-Gly-Ser”, “Gly-Ser-Gly”, “Ser-Gly”, “Gly-Ser” and the nucleotide sequence whose nucleotide sequence encodes a polypeptide. Examples include linkers that encode one or more restriction sites that are particularly suitable for insertion (eg, a “Gly-Ser” linker in which the nucleotide sequence “GGATCC” encodes a BamHI restriction site).

  In a preferred embodiment, the peptide (signal peptide) of the invention consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is the amino terminus of the chimeric polypeptide, ie the first of the polypeptide of interest. Linked at the position before the residue (the initiator Met residue is included in the signal peptide of the present invention).

  In one aspect of this embodiment, the chimeric polypeptide of the invention does not include other signaling peptides to effect secretion of the polypeptide of interest from the host cell into an external medium (eg, culture medium).

  In another preferred aspect of this embodiment, the chimeric polypeptide of the present invention comprises at least one additional signal to immobilize the polypeptide of interest to a cell membrane, particularly the plasma membrane, resulting in presentation on the cell surface. Includes transfer peptides, especially membrane-anchored peptides. The membrane-anchoring peptide is preferably linked at the carboxy terminus of the polypeptide of interest or within the C-terminal portion. The carboxy terminus refers to the last amino acid residue of the polypeptide (residue encoded by the codon prior to the stop codon), while the C-terminal portion refers to the portion of the polypeptide contained in position 3 from the end ( The polypeptide is divided into three parts, an N-terminal part, a central part and a C-terminal part, each assuming one-third of the total polypeptide). Preferably, the C-terminal portion is contained within 100 residues, more preferably within 50 residues immediately before the carboxy terminus of a polypeptide. Membrane-anchored peptides suitable for use in the present invention can be obtained or isolated from cellular or viral proteins, or polypeptides that are naturally bound to the cell membrane. Such membrane-bound proteins are well described in literature publicly available to those skilled in the art (see, for example, Branden and Tooze, 1991, Introduction to Protein Structure p. 202-214, NY Garland). More particularly, rabies glycoprotein, HIV virus envelope glycoprotein and measles virus F protein.

  Preferably, the membrane-immobilized peptide contained in the chimeric polypeptide of the present invention is the membrane-immobilized peptide of the present invention consisting essentially of or consisting of the amino acid sequence represented by any of SEQ ID NOs: 3, 4 or 5. More preferably, the chimeric polypeptide of the present invention comprises a signal peptide consisting of the amino acid sequence shown by SEQ ID NO: 2 and a membrane-fixing peptide consisting of the amino acid sequence shown by SEQ ID NO: 3. Alternatively, the chimeric polypeptide of the present invention comprises a signal peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 and a membrane-fixing peptide consisting of the amino acid sequence shown by SEQ ID NO: 5.

  Since the signal peptides and membrane-anchored peptides described herein are derived from rabies glycoprotein, the polypeptide of interest contained in the chimeric polypeptide of the present invention is preferably heterologous to the rabies virus. As used herein, a polypeptide that is “heterologous to rabies virus” refers to a polypeptide that is not naturally associated with wild-type rabies virus or that is not encoded by the genome of wild-type rabies virus.

  Suitable target polypeptides include, for example, polypeptides having commercial or research value, as well as polypeptides that can provide therapeutic or prophylactic activity in a host organism that exhibits or is suspected of exhibiting a disease or disorder. . For example, the polypeptide of interest is a hormone, cytokine, immunogen, receptor, chaperone, immunoglobulin, enzyme, growth factor, fluorescent protein such as GFP, transporter protein (such as flavodoxin, globin and metallothionein), toxin, suicide gene product , Selected from the group consisting of inhibitors such as structural proteins and protease inhibitors. In the context of the present invention, the polypeptide of interest is preferably selected from the group consisting of an immunogenic polypeptide and an anti-tumor polypeptide.

An “immunogenic” polypeptide refers to a polypeptide that can induce, stimulate, develop or boost the immune system in the host organism in which it is expressed. Such an immune response can be humoral or cellular or both humoral and cellular. A humoral response elicits antibody production against the polypeptide of interest, and a cellular response elicits T helper cells and / or CTL responses and / or stimulation of cytokine production. In general, the immunogenicity of a polypeptide can be assessed either in vitro or in vivo by a variety of assays standard in the art (used to assess the induction and activation of an immune response). For a review of possible technologies, see, for example, the latest edition of Coligan et al, Current Protocols in Immunology; ed J Wiley & Sons Inc, National Institute of Health). For example, detection can be colorimetric, fluorometric or radioactive, and suitable techniques include ELISA, Western blot, radioimmunoassay and immunoprecipitation assay. Cellular immunity is measured by measuring cytokine profiles secreted by activated effector cells, including those derived from CD4 + and CD8 + T cells (eg, quantification of IFNg producing cells by ELIspot). Assay of antigen-specific T lymphocytes in sensitized subjects (eg, peptide-specific lysis in cytotoxicity assays) by activation status determination (eg, conventional T cell proliferation assay by [ ³ H] thymidine incorporation) This can be done. The immunogenicity of a polypeptide can also be assessed in a suitable animal model by ELIspot, tetramer-based analytical techniques or other standard techniques for analysis of T cell immunity. Suitable immunogenic polypeptides are those derived from hepatitis B virus (HBV) (eg, S, preS2 or preS1-polypeptide as described in EP414374; EP304578 or EP198474); hepatitis C virus (HCV ) Obtained from (eg, Core (C), envelope glycoprotein E1, E2, nonstructural polypeptide NS2, NS3, NS4 or NS5 or any combination thereof); obtained from human immunodeficiency virus (HIV) ( For example, from gp120 or gp160) and papilloma virus.

  The papillomavirus immunogenic polypeptides included in the chimeric polypeptides of the invention can be early, late, or any combination thereof. Early papillomavirus polypeptides include E1, E2, E4, E5, E6 and E7, and the late polypeptide is L1 or L2. Preferably HPV-16, HPV-18, HPV-30, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, Obtained from a papilloma virus selected from the group consisting of HPV-59, HPV-66, HPV-68, HPV-70 and HPV-85. Nucleotide sequences of several papillomavirus genomes and amino acid sequences of the encoded polypeptides are available in specialized data banks. For summary information, the HPV-16 genome is as accession numbers NC_01526 and K02718; HPV-18 is NC_001357 and X05015; HPV-31 is as J04353; HPV-33 is as M12732; HPV-35 is as NC_001529; HPV-39 Is as NC_001535; HPV-45 is as X74479; HPV-51 is as NC_001533; HPV-52 is as NC_001592; HPV-56 is as X74483; HPV-58 is as D90400; HPV-59 is as NC_001635; HPV-68 is As X67160 and M73258; HPV-70 is described as U21941; and HPV-85 is described in Genbank as AF131950 That. However, the invention is not limited to these exemplary sequences. Indeed, the natural genetic variations found between different papilloma virus isolates, as well as those described in literature available to those skilled in the art (eg, Yasugi et al. 1997 describing modified E1 polypeptides). , J. Virol 71, 5942-51; Sakai et al., 1996, J. Virol. 70, 1602-1611 describing modified E2 polypeptides and modified E6 and E7 polypeptides suitable for use in the present invention Non-naturally occurring modifications such as WO99 / 03885, which describe) are also included within the scope of the present invention.

  An “anti-tumor” polypeptide refers to a polypeptide that can provide an inhibition or a net decrease in the expansion of tumor cells. The anti-tumor properties of a polypeptide can be measured by a reduction in actual tumor size over a period of time in an appropriate animal model or in a treated subject. To assess tumor size, radiological methods (eg, single photon and positron emission computed tomography; generally “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer-Verlag, New York 1986), methods using conventional imaging reagents (eg, gallium citrate-67), immunological methods (eg, radiolabeled monoclonal antibodies directed to specific tumor markers) and ultrasound ("Ultrasonic Differential"). Various methods can be used, including "Diagnosis of Tumors", Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984). Alternatively, the anti-tumor properties of a polypeptide can be measured based on a decrease in the presence of a tumor marker. Examples include PSA for detection of prostate cancer, and CEA for detection of colorectal cancer and certain types of breast cancer. Furthermore, the anti-tumor properties of a polypeptide can also be measured in a suitable animal model, for example using a mouse injected with a representative human cancer cell line. After the palpable tumor develops, the mice are injected with the vector of the invention and then monitored for decreased tumor growth rate and increased survival. In addition, various in vitro methods can be used to estimate tumor inhibition in vivo. Suitable anti-tumor polypeptides include MUC-I (WO92 / 07000; Acres et al, 2005, Exp. Rev. Vaccines 4 (4)), BRCA-I, BRCA-2 (Palma et al., 2006, Critical Reviews in Oncology / haematology 27, 1-23), carcinoembryonic antigen CEA (Conroy et al., 1995, Gene Ther; 2, 59-65), MAGE (WO 99/40188; De Plaen et al., 1994, Immunogenetics) 40, 360-369), MART-1, gp 100 (Bakker et al., 1994, J. Exp. Med. 179, 1005-9), PRAME, BAGE, Lage (also known as NY Eos 1) SAGE, HAGE (WO 99/53061), GAGE (Robbins and Kawakami, 1996. Current Opinions in Immunol. 8, 628-36) and prostate specific antigen (PSA) (Ferguson, et al., 1999, Proc. Natl. Acad. Sci USA. 96, 3114-9; WO 98/12302, WO 98/2017 and WO 00/04149) and Examples include tumor-associated antigens (TAA) such as virus polypeptides derived from viruses having tumor-inducing ability.

  The invention also provides an isolated nucleic acid molecule comprising, consisting essentially of, or consisting of at least a nucleotide sequence encoding a peptide of the invention or a chimeric polypeptide of the invention.

  In a preferred embodiment, the present invention provides an isolated nucleic acid molecule comprising at least one of the nucleotide sequences set forth in any of SEQ ID NOs: 11-28.

  More specifically, the nucleotide sequences of SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 15 encode a signal peptide having the amino acid sequence of SEQ ID NO: 1; the nucleotide sequence of SEQ ID NO: 12 is the amino acid sequence of SEQ ID NO: 2 The nucleotide sequence of SEQ ID NO: 13 encodes the signal peptide present in the rabies glycoprotein of the Nishigahara strain (disclosed in UniProt as accession number S72465); the nucleotide sequence of SEQ ID NO: 16 Encodes a signal peptide present in the rabies glycoprotein of the ERA strain (disclosed in UniProt as accession number M38452).

  The nucleotide sequence shown in SEQ ID NO: 17 and SEQ ID NO: 22 encodes a native membrane anchored peptide present in the rabies glycoprotein of the PG strain (disclosed to UniProt as accession number ay009097); shown in SEQ ID NO: 18 The nucleotide sequence encodes a membrane anchored peptide having the amino acid sequence of SEQ ID NO: 3; the nucleotide sequence shown in SEQ ID NO: 19 encodes a membrane anchored peptide having the amino acid sequence of SEQ ID NO: 4; The nucleotide sequence shown encodes a membrane anchored peptide having the amino acid sequence of SEQ ID NO: 5.

  The nucleotide sequence of SEQ ID NO: 23 (M38452, v1) comprises the nucleotide sequence disclosed in SEQ ID NO: 11 linked to the nucleotide sequence of SEQ ID NO: 17 via a BamHI linker; Comprises the nucleotide sequence disclosed in SEQ ID NO: 12 linked to the nucleotide sequence of SEQ ID NO: 18 via a BamHI linker; the nucleotide sequence of SEQ ID NO: 25 is of SEQ ID NO: 19 via a BamHI linker Comprising the nucleotide sequence disclosed in SEQ ID NO: 13 linked to the nucleotide sequence; the nucleotide sequence of SEQ ID NO: 26 is linked to SEQ ID NO: 14 linked to the nucleotide sequence of SEQ ID NO: 20 via a BamHI linker. The disclosed nucleotide sequence The nucleotide sequence of SEQ ID NO: 27 comprises the nucleotide sequence disclosed in SEQ ID NO: 15 linked to the nucleotide sequence of SEQ ID NO: 21 via a BamHI linker; and the nucleotide sequence of SEQ ID NO: 28 Comprises the nucleotide sequence disclosed in SEQ ID NO: 16 linked to the nucleotide sequence of SEQ ID NO: 22 via a BamHI linker.

  Further, the nucleotide sequences of SEQ ID NOs: 23-28 reduce the percentage of homology to each other to less than 75% as shown in the appended examples section, and thus secretion of multiple polypeptides of interest and / or A multigene expression vector for enabling membrane fixation is manipulated so that at least two of them can be used together. The combined use of the nucleotide sequences of the invention is advantageous when multiple polypeptides need to be expressed from a single vector and secreted and / or transported to the cell membrane. The use of the nucleotide sequences of the present invention can reduce the risk of homologous recombination events that can occur when homologous sequences are present in the same vector. In the art, natural rabies signaling sequences show high homology between them, and such homology prevents their combination, adversely affects vector stability, and is unsuitable for human use. May bring the original stock.

  Reduction of the percentage homology between two nucleic acid molecules of the invention is generally done by modifying the codon usage pattern. At the first point in time, the sequence alignment between the nucleic acid molecules before modification can be used to reveal the homologous portion. The genetic code degeneracy is then used to reduce the homology percentage of such homologous parts to less than 75%. Methionine and tryptophan residues are each encoded by a unique nucleic acid triplet (ie, a codon), but various codons can be used to encode the other 18 amino acids (genetic code degeneracy). . Modification of the codon usage pattern is generally done by replacing one or more “natural” codons with another codon. For example, if the AGA codon that encodes Arg is replaced with a CGC codon that encodes Arg, two of the three codon positions lose homology. It is not necessary to degenerate all natural codons since homology is sufficiently reduced by partial substitution.

  Furthermore, the disclosed nucleotide sequences can be further modified by those skilled in the art. For example, the first codon encoding the initiator Met residue present at the N-terminus of the signal peptide and / or the last codon encoding the TGA stop codon present at the C-terminus of the membrane-anchored peptide may be It may be deleted as long as it has any regulatory codons. In addition, the BamHI linker present at the junction between the signal sequence and the membrane anchoring sequence can be replaced with another linker that contains further restriction sites suitable for cloning the nucleotide sequence encoding the polypeptide.

  In a preferred embodiment, the nucleotide sequence encoding the polypeptide of interest comprises a nucleotide sequence encoding a signal peptide (defined in SEQ ID NOs: 11 to 16) and a nucleotide sequence encoding a membrane-anchored peptide in the nucleic acid molecule of the present invention ( (Defined by SEQ ID NOs: 17 to 22). More preferably, it is inserted within a BamHI linker between approximately number 70 and number 76 of SEQ ID NOs: 23-28.

  In another preferred embodiment, the nucleic acid molecule of the invention is placed under the control of appropriate regulatory elements to ensure its expression in a given host cell or organism. As used herein, “regulatory sequence” refers to a given host comprising replication, doubling, transcription, splicing, translation, stability and / or transport of one of its nucleic acids or derivatives thereof (ie, mRNA) into the host cell. Any sequence that allows, contributes to or regulates the expression of a nucleic acid molecule in a cell. Such regulatory sequences are well known in the art (see, eg, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego). One skilled in the art can select regulatory sequences based on factors such as the type of vector, the host cell, the desired level of expression and the like.

  Promoters are particularly important, and the present invention relates to constitutive promoters that express nucleic acid molecules in many types of host cells, and only in certain host cells (eg, tissue-specific regulatory sequences) or specific events or exogenous Includes the use of those expressed in response to sex factors (eg, by temperature, nutritional additives, hormones or other ligands). Suitable promoters for constitutive expression in eukaryotic systems include SV40 promoter, cytomegalovirus (CMV) immediate early promoter or enhancer (Boshart et al, 1985, Cell 41, 521-530), adenovirus early and late Promoters, viral promoters such as herpes simplex virus (HSV) -1 thymidine kinase (TK) promoter and retroviral long terminal repeats (eg, MoMuLV and Rous sarcoma virus (RSV) LTR), and phosphoglycerokinase (PGK) Examples include cellular promoters such as promoters (Hitzeman et al., 1983, Science 219, 620-625; Adra et al., 1987, Gene 60, 65-74). Suitable promoters useful for driving the expression of nucleic acid molecules in poxvirus vectors include the vaccinia virus 7.5K, H5R, TK, p28, p11 or K1L promoter. Alternatively, described in Chakrabarti et al. (1997, Biotechniques 23, 1094-1097), Hammond et al. (1997, J. Virological Methods 66, 135-138) and Kumar and Boyle (1990, Virology 179, 151-158) Synthetic promoters such as those that have been described, and chimeric promoters between the early and late poxvirus promoters.

  Inducible promoters are regulated by exogenously supplied compounds and include, but are not limited to, zinc-induced metallothionein (MT) promoter (Mc Ivor et al., 1987, Mol. Cell Biol. 7, 838-848), Dexamethasone (Dex) induced mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system (WO 98/10088), ecdysone insect promoter (No et al., 1996, Proc. Natl. Acad. Sci. USA 93, 3346-3351), Tetracycline repressible promoter (Gossen et al., 1992, Proc. Natl. Acad. Sci. USA 89, 5547-5551), tetracycline inducible promoter (Kim et al., 1995, J. Virol. 69, 2565-2573), RU486 Inducible promoter (Wang et al., 1997, Nat. Biotech. 15, 239-243 and Wang et al., 1997, Gene Ther. 4, 432-441), rapamycin inducible promoter (Magari et al., 1997, J. Clin. Invest. 100, 2865-2872) and large intestine Fungal lac, TRP and TAC promoters are included.

  Regulatory sequences in use in connection with the present invention can also be tissue specific so as to drive the expression of the nucleic acid molecule in the specific tissue where a therapeutic benefit is desired. Suitable promoters may be taken from genes that are preferentially expressed in tumor cells. Such genes can be identified, for example, by display and comparative genomic hybridization (see, eg, US Pat. Nos. 5,759,776 and 5,776,683).

  Those skilled in the art will further understand that regulatory elements that control the expression of nucleic acid molecules may further direct, regulate and / or terminate transcription (eg, poly A transcription termination sequences), mRNA transport (eg, nuclear localization signal sequences), processing ( For example, additional elements suitable for splicing signals), stability (eg, introns and non-coding 5 ′ and 3 ′ sequences) and translation (eg, tripartite leader sequence, ribosome binding site, Shine-Dalgamo sequence, etc.) It can be seen that it may be contained in a cell or organism.

  The invention also provides a vector for expressing one or more chimeric polypeptides of the invention. Advantageously, the vector of the invention comprises one or more nucleic acid molecules as described herein, particularly preferably 2, 3 or even 4 nucleic acid molecules. Desirably, each nucleic acid molecule inserted into the vector of the present invention differs in at least the signal peptide, membrane-immobilized peptide and / or polypeptide of interest encoded by it. Preferably, the vectors of the present invention have 2, 3, 4 or even more signal and membrane-anchored peptide-encoding nucleotide sequences (for example from SEQ ID NOs: 23-28) showing a percentage of homology to each other of less than 75%. Selected from the group consisting of: More preferably, the vector of the present invention can express the nucleotide sequence of SEQ ID NO: 24 for allowing the expression of the first polypeptide of interest on the cell membrane and the expression of the second polypeptide of interest on the cell membrane. Comprising the nucleotide sequence of SEQ ID NO: 28.

  A variety of vector systems can be used to express the chimeric polypeptides of the present invention and are suitable for prokaryotic cells (eg, E. coli or Bacillus subtilis), bacteriophage, plasmid or cosmid vectors. A yeast expression vector suitable for yeast host cells (eg, Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris); suitable for insect host cells (eg, Sf9 cells); Viral expression vectors (eg, baculovirus); viral expression vectors (eg, cauliflower mosaic virus CaMV; tobacco mosaic virus TMV) or plasmid vectors suitable for plant cells (eg, Ti plasmid); or higher eukaryotic host cells or organisms Suitable plastic Or it contains a virus vector.

  Suitable vectors for use in prokaryotic systems include, but are not limited to, pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), p Poly (Lathe et al, 1987, Gene 57, 193-201), pTrc (Amann et al., 1988, Gene 69, 301-315); pET 11d (Studier et al., 1990, Gene Expression Technology: Methods in Enzymology 185, 60-89); pIN (Inouye et al., 1985, Nucleic Acids Res. 13, 3101-3109; Van Heeke et al., 1989, J. Biol. Chem. 264, 5503-5509); and the nucleic acid molecules of the invention with glutathione S-transferase (GST) And pGEX vectors that can be expressed in a fusion state.

  Suitable vectors (eg, S. cerevisiae) for expression in yeast include, but are not limited to, pYepSec1 (Baldari et al., 1987, EMBO J. 6, 229-234), pMFa (Kujan et al , 1982, Cell 30, 933-943), pJRY88 (Schultz et al., 1987, Gene 54, 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.) And pTEF-MF (Dualsystems Biotech Product code: P03303).

  Suitable vectors for expression in eukaryotic host cells or organisms are viral or non-viral (eg, plasmid DNA). Suitable plasmid vectors include, but are not limited to, pREP4, pCEP4 (Invitrogene), pCI (Promega), pCDM8 (Seed, 1987, Nature 329, 840), pMT2PC (Kaufman et al., 1987, EMBO J 6, 187-195), pVAX and pgWiz (Gene Therapy System Inc; Himoudi et al, 2002, J. Virol. 76, 12735-12746). In the present specification, the “viral vector” is used according to the meaning recognized in the field. A viral vector can be a complete viral genome, a portion thereof, or a modified viral genome as described below, as well as produced viral particles (eg, viral vectors packaged in a viral capsid to produce infectious viral particles). And any vector containing at least one element of viral origin. The viral vector of the present invention may be capable of replication or may be genetically disabled and may be replication defective or replication defective. As used herein, “replication ability” includes replication-selective and conditionally replicating viral vectors that are engineered to replicate well or selectively in a particular host cell (eg, tumor cell). Viral vectors can be obtained from a variety of different viruses, in particular from viruses selected from the group consisting of retroviruses, adenoviruses, adeno-associated viruses (AAV), poxviruses, herpesviruses, measles viruses and foamy viruses. .

  In one embodiment, the vector of the invention is an adenoviral vector (for review see "Adenoviral vectors for gene therapy", 2002, Ed D. Curiel and J. Douglas, Academic Press). It can be derived from either human or animal adenovirus. Any serotype and subgroup can be used in the present invention. More particularly, subgroup A (eg, serotypes 12, 18, and 31), subgroup B (eg, serotypes 3, 7, 11, 14, 16, 21, 34 and 35), subgroup C (eg, Serotypes 1, 2, 5, and 6), subgroup D (eg, serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39 and 42-47) ), Subgroup E (serotype 4) and subgroup F (serotypes 40 and 41). Particularly preferred are human adenovirus 2 (Ad2), 5 (Ad5), 6 (Ad6), 11 (Ad11), 24 (Ad24) and 35 (Ad35). Such adenoviruses are available from the American Type Culture Collection (ATCC, Rockville, Md.) And are the subject of many publications that describe their sequence, organization, and production methods. Can be applied (eg, US Pat. Nos. 6,133,028, 6,110,735, WO 02/40665, WO 00/50573, EP 1016711, Vogels et al, 2003, J. Virol). 77, 8263-8271).

  The adenoviral vector of the present invention can have replication ability. One skilled in the art can readily obtain many examples of replicating adenoviral vectors (eg, Hernandez-Alcoceba et al., 2000, Human Gene Ther. 11, 2009-2024; Nemunaitis et al. , 2001, Gene Ther. 8, 746-759; Alemany et al., 2000, Nature Biotechnology 18, 723-727; WO 00/24408; US Pat. No. 5,998,205, WO 99/25860, US Pat. 698,443, WO00 / 46355, WO00 / 15820 and WO01 / 36650).

  Alternatively, the adenoviral vector of the present invention may be replication-defective (see, for example, WO94 / 28152). A preferred replication-defective adenoviral vector is E1 defective (eg, US Pat. Nos. 6,136,594 and 6,013,638), and deletion of E1 is approximately number 459-3328 or approximately 459. (See the sequence of the human type 5 adenovirus disclosed in GeneBank as accession number M73260 and also in Chroboczek et al, 1992, Virol. 186, 280-285). Cloning ability and safety can be achieved with the adenovirus genome (eg, in the non-essential E3 region or other essential E2, E4 regions as described in Lusky et al., 1998, J. Virol 72, 2022-2032). Further improvements can be made by deleting additional portions.

  The nucleic acid molecule of the present invention can be inserted at any position of the adenoviral vector as described in Chartier et al. (1996, J. Virol. 70, 4805-4810). They can be arranged in a sense orientation and / or an antisense orientation relative to the original transcription direction. For example, it can be inserted in place of the E1 region or in place of the E3 region.

  In another embodiment, the vector of the invention is a poxvirus vector (see, eg, Cox et al. In “Viruses in Human Gene Therapy” Ed J. M. Hos, Carolina Academic Press). It may be obtained from any member of the Poxviridae family, in particular canarypox virus (eg ALVAC as described in WO 95/27780), fowlpox virus (eg Paoletti et al., 1995, Dev Biol. Stand. 84, 159-163) or a vaccinia virus, preferably a vaccinia virus. Suitable vaccinia viruses are Copenhagen strains (Goebel et al., 1990, Virol. 179, 247-266 and 517-563; Johnson et al., 1993, Virol. 196, 381-401), Weiss strain, NYVAC (WO92 / 15672 and Tartaglia et al., 1992, Virology 188, 217-232) and a highly attenuated modified Ankara (MVA) strain (Mayr et al., 1975, Infection 3, 6-16) it can. Such vectors and production methods are described in a number of literature available to those skilled in the art (eg, Paul et al., 2002, Cancer gene Ther. 9, 470-477; Piccini et al., 1987, Methods of Enzymology 153, 545-563; U.S. Pat. Nos. 4,769,330; 4,772,848; 4,603,112; 5,100,587 and 5,179. 993). The nucleic acid molecules of the invention are preferably inserted into a nonessential locus of the poxvirus genome so that the recombinant poxvirus remains viable and infectious. A non-essential region is a non-coding intergenic region or any gene whose inactivation or deletion does not significantly impair viral growth, replication or infection. For example, by using a helper cell line having a complementary sequence corresponding to that deleted in the poxvirus genome, the essential virus can be used as long as the missing function is supplied in trans during the production of virus particles. Insertion at the locus is also conceivable.

  When using Copenhagen vaccinia virus, the nucleic acid molecule is preferably inserted into the thymidine kinase gene (tk) (Hruby et al, 1983, Proc. Natl. Acad. Sci USA 80, 3411-3415; Weir et al., 1983 , J. Virol. 46, 530-537). However, for example, the hemagglutinin gene (Guo et al., 1989, J. Virol. 63, 4189-4198), K1L locus, u gene (Zhou et al., 1990, J. Gen. Virol. 71, 2185). -2190) or various natural or engineered deletions (Altenburger et al., 1989, Archives Virol. 105, 15-27; Moss et al. 1981, J. Virol. 40, 387-395; Panicali et al., 1981, J. Virol. 37, 1000-1010; Perkus et al, 1989, J. Virol. 63, 3829-3836; Perkus et al, 1990, Virol. 179, 276-286; Perkus et al, 1991 , Virol. 180, 406-410) and other insertion sites such as the left end of the vaccinia virus genome are also suitable.

  When using MVA, the nucleic acid molecule can be inserted into any of the identified deletions I-VII present in the MVA genome (Antoine et al., 1998, Virology 244, 365-396) as well as the D4R locus. Although preferred, insertions in deletions II and / or III are preferred (Meyer et al., 1991, J. Gen. Virol. 72, 1031-1038; Sutter et al., 1994, Vaccine 12, 1032-1040).

  When fowlpox virus is used, insertion into the thymidine kinase gene is envisaged, but the nucleic acid molecule is preferably introduced into the intergenic region located between ORFs 7 and 9 (see, eg, EP 314 569 and US Pat. No. 5, 180,675).

  Furthermore, the vectors of the invention may contain one or more additional elements that allow the maintenance, amplification or expression of the nucleic acid molecule in a given host cell or organism. Such additional elements include, but are not limited to, marker genes to facilitate identification and isolation of recombinant host cells (eg, by supplementing cell auxotrophy or by antibiotics). Resistance), stabilizing elements (eg, the cer sequence described in Summers and Sherrat, 1984, Cell 36, 1097-1103 and the DAP described in US Pat. No. 5,198,343) and integration elements ( For example, LTR viral sequences and transposons).

  The present invention also provides infectious viral particles comprising the above-described nucleic acid molecules or vectors. Here, the various methods known for the production of infectious virus particles are not described in detail. In general, such viral particles are (a) introducing a viral vector into an appropriate cell line, (b) culturing the cell line under conditions suitable to allow production of the infectious viral particle. The infectious virus particles produced are recovered from the cell line culture and, if desired, produced by a method comprising the step of purifying the recovered infectious virus particles.

  If the viral vector is defective, infectious particles are usually produced in supplemental cell lines that supply non-functional viral genes in trans. For example, 293 cells (Graham et al, 1997, J. Gen. Virol. 36, 59-72), PER-C6 cells (Fallaux et al. , 1998, Human Gene Ther. 9, 1909-1917) and HER96 cells. Suitable cells for amplifying the poxvirus vector are avian cells, most preferably primary chicken embryo fibroblasts (CEF) prepared from chicken embryos obtained from fertilized eggs. These production cells can be cultured in conventional fermentation reactors, flasks and petri plates under conditions of appropriate temperature, pH and oxygen content. Infectious virus particles can be recovered from the culture supernatant or the lysed cells. They are according to standard techniques (eg chromatography, ultracentrifugation as described in WO96 / 27677, WO98 / 00524, WO98 / 22588, WO98 / 26048, WO00 / 40702, EP1016700 and WO00 / 50573). Further purification is possible.

  The present invention also encompasses viral vectors that have been modified to be able to preferentially target specific host cells. Targeting vectors are characterized by ligands that can recognize and bind to cells and surface exposed components such as cell-specific markers (eg, HPV infected cells), tissue-specific markers or tumor-specific markers on their surface. It exists. This ligand can generally be inserted into a polypeptide (eg, adenovirus fiber, penton, pIX described in WO94 / 10323 and WO02 / 96939 or the vaccinia p14 gene product described in EP1146125) present on the surface of the vector.

  The invention also relates to a host cell comprising a nucleic acid molecule, vector or infectious viral particle as described above.

  Host cells in the context of the present invention include prokaryotic cells (eg, Escherichia coli, Bacillus, Listeria), yeast (eg, Saccharomyces cerevisiae), Saccharomyces pombe or Pichia pastoris ( Lower eukaryotic cells such as Pichia pastoris)), and other eukaryotic cells such as insect cells, plant cells and higher eukaryotic cells, including mammalian cells (eg, human or non-human cells) are particularly preferred. Representative examples of suitable host cells include, but are not limited to, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell cat kidney cell line), CV- 1 cell (African monkey kidney cell line), COS (eg COS-7) cell, Chinese hamster ovary (CHO) cell, mouse NIH / 3T3 cell, HeLa cell and Vero cell. The term “host cell” also encompasses supplemental cells capable of complementing at least one defective function of a replication defective vector (eg, an adenoviral vector) of the present invention, such as those cited above.

Methods for introducing nucleic acid molecules, vectors or infectious particles into isolated host cells are routine in the art, such as microinjection (Capechi et al., 1980, Cell 22, 479-488), CaPO ₄ mediated transfection (Chen and Okayama, 1987, Mo Cell Biol. 7, 2745-2752), DEAE- dextran-mediated transfection, electroporation (Chu et al, 1987, Nucleic Acid Res. 15, 1311-1326) Lipofection / liposome fusion (Feigner et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7413-7417), particle bombardment (Yang et al., 1990, Proc. Natl. Acad. Sci. USA 87, 9568-9572), gene gun, transduction, viral infection and direct administration to the host organism by various means.

  Host cells can be used to produce the polypeptide of interest encoded by the nucleic acid molecules, vectors or infectious particles of the invention by recombinant means. Such techniques are well known in the art (eg, Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002; and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press See the latest issue).

  In another aspect of the invention, the chimeric polypeptide, vector, infectious viral particle or host cell (also referred to herein as an “effective agent”) or any combination thereof is included in the composition. Advantageously, the composition is a pharmaceutical composition comprising a therapeutically effective amount of an active agent and a pharmaceutically acceptable vehicle. As used herein, a “therapeutically effective amount” is a dose sufficient to alleviate one or more symptoms normally associated with a disease or condition that is desired to be treated or prevented in a host organism. For prophylactic use, the term refers to a dose sufficient to avoid or delay the establishment of a disease or condition in the host organism. For example, a therapeutically effective amount is an amount sufficient to induce or enhance an immune response in the treated organism, or reduce or ameliorate, stabilize, reverse, slow or delay progression (eg, lesions) Or a reduction or regression of tumor size, reversal of viral infection).

As used herein, “pharmaceutically acceptable vehicle” refers to carriers, solvents, diluents, excipients, adjuvants, dispersion media, coating agents, antibacterial and antifungal agents, absorption delaying agents and the like suitable for pharmaceutical administration. Any one or all of the above shall be included. Desirably, the compositions of the present invention comprise one or more carriers and / or diluents that are non-toxic at the dosages and concentrations employed. Such carriers and / or diluents are preferably selected from those commonly used to formulate compositions in either unit dosage forms or multiple dosage forms for systemic or mucosal administration. A suitable carrier can be a dispersion medium containing a solvent such as water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), vegetable oils, or suitable mixtures thereof. The diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples of suitable diluents include sterile water, saline (eg, sodium chloride), Ringer's solution, glucose, trehalose or saccharose solution, Hank's solution and other physiologically balanced salt solutions (eg, Remington: The Science and the Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins (see the latest issue). The pH of the composition of the present invention is adjusted as appropriate, preferably physiological pH or slightly basic pH (between about pH 7.5 and about pH 9, so as to be suitable for human or animal use. pH 8 to 8.5 is particularly preferred). Suitable buffers include phosphate buffer (eg, PBS), bicarbonate buffer and / or Tris buffer. A particularly preferred composition (especially when the active agent is an adenoviral vector) is formulated in 1M saccharose, 150 mM NaCl, 1 mM MgCl ₂ , 54 mg / l Tween 80, 10 mM Tris pH 8.5. Another preferred composition is formulated in 10 mg / ml mannitol, 1 mg / ml HSA, 20 mM Tris, pH 7.2 and 150 mM NaCl. Such formulations are stable for at least two months at either freezing temperatures (eg -70 ° C, -40 ° C, -20 ° C) or refrigeration temperatures (eg 4 ° C). It is particularly suitable for maintaining.

  The composition also includes, for example, modification or maintenance of pH, osmotic pressure, viscosity, clarity, color, sterility, stability, dissolution rate of the formulation, release or absorption into a human or animal subject. One or more pharmaceutically acceptable excipients can be included to provide the desired pharmacological or pharmacokinetic properties.

  Furthermore, the compositions of the present invention can include one or more adjuvants suitable for systemic or mucosal administration in humans. “Adjuvant” means a compound that has the ability to enhance an immune response to a particular antigen. Adjuvants can be delivered near the antigenic site. Enhancement of humoral immunity is generally manifested by a significant increase (usually more than 10 times) of the titer of antibodies raised against that antigen. Enhancement of cellular immunity can be measured, for example, by positive skin tests, cytotoxic T cell assays, ELIspot assays for IFNg or IL-2. Preferably, adjuvants in use in the present invention are capable of stimulating immunity against an active agent, particularly via toll-like receptors (TLRs), such as TLR-7, TLR-8 and TLR-9. Representative examples of useful adjuvants include, but are not limited to, mineral oil emulsions such as alum, Freund's complete and incomplete adjuvant (IFA), lipopolysaccharides or derivatives thereof (Ribi et al., 1986, Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p407-19); saponins such as QS21 (Sumino et al., 1998, J. Virol. 72, 4931-9; WO 98/56415); Imiquimod (Suader, 2000 , J. Am Acad Dermatol. 43, S6-S11), 1H-imidazo (4,5-c) quinolone-4-amine derivatives (Aldara ™) and related compounds (Smorlesi, 2005, Gene Ther. 12, 1324 -32) imidazoquinoline compounds; cytosine phosphate guanosine such as CpG (Chu et al., 1997, J. Exp. Med. 186: 1623; Tritel et al., 2003, J. Immunol. 171: 2358-2547) Oligodeoxynucleotides; and IC-31 (Kritsch et al., 2005, J. And cationic peptides such as Chromatogr Anal. Technol Biomed Life Sci 822, 263-70).

  The compositions of the present invention can be in various forms, for example, a solid (eg, a dry powder form or a lyophilized form), or a liquid (eg, an aqueous solution). A solid composition of the active agent and any additional desired ingredients can be obtained by vacuum drying and lyophilizing from the solution previously sterilized by filtration. If desired, it can be stored in a sterile ampoule that is simply reconstituted by adding a suitable vehicle prior to use.

  The chimeric polypeptide, nucleic acid molecule, vector, infectious particle or composition of the invention can be administered by a variety of modes of administration including systemic administration, topical administration and mucosal administration. Systemic administration may be performed by any means such as subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intravascular, intraarterial injection. The injection can be made with a conventional syringe and needle, or any other suitable device available in the art. Mucosal administration can be by oral, nasal, intratracheal, intrapulmonary, intravaginal or rectal route. Topical administration can be performed using transdermal means (eg, patches, etc.). Intramuscular or subcutaneous administration is particularly preferred when using viral vectors and infectious particles as active agents.

The appropriate dose depends on the pharmacokinetic properties of the particular active agent, the age, health and weight of the subject, the symptoms to be treated, the nature and extent of symptoms, the type of concurrent treatment, the frequency of treatment, the need for prevention or treatment and It may vary depending on known factors such as the desired effect. The dose can also be calculated according to the particular route chosen. Further refinement of the calculations necessary to determine the appropriate dose for treatment is usually performed by the physician in the context of the relevant situation. As a general guide, a suitable dose of adenoviral particles is about 10 ⁵ to about 10 ¹³ iu (infectious units), desirably about 10 ⁷ to about 10 ¹² iu, preferably about 10 ⁸ to about 10 ¹¹ iu. And various. Suitable doses of vaccinia virus particles vary from about 10 ⁴ to about 10 ¹⁰ pfu (plaque-forming particles), desirably from about 10 ⁵ to about 10 ⁹ pfu, preferably from about 10 ⁶ to about 5 × 10 ⁸ pfu. . The vector plasmid can be administered at a dose between 10 μg and 20 mg, preferably between 100 μg and 2 mg, and the chimeric polypeptide can be administered between 05 μg and 5 g, preferably between 5 μg and 500 mg.

In addition, administration can be performed in a single dose, or alternatively in multiple doses according to standard protocols, dosing regimens and dosing schedules over hours, days and / or weeks. Administration can also be by bolus injection or continuous infusion. For example, after three consecutive doses every other week, further doses can be given at least once within 4-6 months after the first series. As a general guide, when using an MVA vector as a therapeutic agent, the preferred route of administration is subcutaneous with a dose of MVA particles comprised between 10 ^{6 and} 5 × 10 ⁸ pfu.

  The chimeric polypeptides, nucleic acid molecules, vectors, infectious particles, host cells or compositions of the invention can be used in methods of treating or preventing various diseases and conditions, including genetic diseases, congenital diseases and acquired diseases. is there. The invention also relates to the use of a chimeric polypeptide, vector, infectious particle, host cell or composition of the invention for the manufacture of a medicament intended to treat or prevent such diseases. It is particularly suitable for treating or preventing infectious diseases (eg viral and / or bacterial infections), cancer and immunodeficiency diseases. “Cancer” includes any cancerous condition caused by unwanted cell proliferation, including chronic or localized tumors, metastases, cancerous polyps and preneoplastic lesions (eg, neoplasms). Cancers contemplated for the present invention include, but are not limited to, glioblastoma, sarcoma, melanoma, mastocytoma, carcinoma as well as breast cancer, prostate cancer, testicular cancer, ovarian cancer, endometrial cancer, child Cancer of the cervix (especially related to papilloma virus infection), lung cancer (eg large cell cancer, small cell cancer, squamous cell carcinoma and adenocarcinoma), kidney cancer, bladder cancer, liver cancer, colon cancer, anal cancer, pancreatic cancer Gastric cancer, gastrointestinal cancer, oral cancer, laryngeal cancer, brain and CNS cancer, skin cancer (eg, melanoma and non-melanoma), blood cancer (especially lymphoma, leukemia if they develop in solid mass), bone Cancer, retinoblastoma and thyroid cancer. Infectious diseases include those caused by any pathogenic microorganism, such as a virus. Infectious diseases contemplated by the present invention include those associated with infection by hepatitis viruses (eg, hepatitis A, B or C). Infectious diseases contemplated by the present invention include those associated with papillomavirus (eg, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52), Such diseases include persistent infections, pre-malignant lesions (eg, low grade, intermediate grade or high grade cervical intraepithelial neoplasia) and malignant lesions (eg cervical cancer).

  The present invention also includes a) introducing the vector or infectious viral particle of the present invention into a host cell, b) culturing the transformed host cell under conditions that allow expression of the polypeptide of interest, and c) A method for recombinant production of a polypeptide comprising recovering the polypeptide from the culture and optionally purifying it.

  In one embodiment, the polypeptide is produced to be secreted into the extracellular medium according to the methods of the invention. This polypeptide is preferably included in a chimeric polypeptide comprising a signal peptide as described herein, whereby the signal peptide is cleaved from the chimeric polypeptide and the polypeptide is recovered from the culture medium.

  In another embodiment, the polypeptide is produced to be immobilized on a cell membrane according to the method of the invention, with attachment to the outer surface of the plasma membrane being particularly preferred. Preferably, the polypeptide is immobilized such that it can interact with other molecules present on other cell surfaces or extracellular media. The polypeptide is preferably included in a chimeric polypeptide comprising a signal peptide and a membrane peptide as described herein, whereby the signal peptide is cleaved from the chimeric polypeptide and the polypeptide is recovered from the cultured cells. .

  The present invention also encodes the polypeptide with (a) a first nucleotide sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. A step of producing a nucleic acid molecule by linking a second nucleotide sequence (the nucleic acid molecule comprises a signal peptide encoded by the first nucleotide sequence and a target polypeptide in the N-terminal to C-terminal direction) (B) the step of inserting the obtained nucleic acid molecule into an expression vector, and (c) the step of introducing the expression vector into a host cell or organism, and the target polypeptide outside the cell. Provides a method of expressing. As described above, the signal peptide is cleaved from the chimeric polypeptide upon secretion, and then the target polypeptide is released out of the cell. It can be recovered from the culture medium and, if desired, purified using routine methods in the art.

  The present invention also provides (a) a polypeptide of interest encoded by a first nucleotide sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28. Inserting a second nucleotide sequence to be inserted into any position from the nucleotide at position 70 to the nucleotide at position 76 of the first nucleotide sequence (the nucleic acid molecule is A signal peptide encoded by the first nucleotide sequence, a target polypeptide encoded by the second nucleic acid, and a membrane-anchored peptide encoded by the first nucleotide sequence in the N-terminal to C-terminal direction. And (b) inserting the resulting nucleic acid molecule into an expression vector; and ) The expression vector comprising the step of introducing into a host cell or organism, provides a method of expressing the immobilized polypeptide on the surface of the plasma membrane. As described above, the signal peptide is cleaved from the chimeric polypeptide and expressed so that the polypeptide with the membrane-anchored peptide can interact with other molecules present on other cell surfaces or extracellular media. Presented on the surface of the host cell.

  The nucleic acids, vectors and polypeptides of the invention described herein are described, for example, in Sambrook et al. (2001, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY), Ausubel et al. (1994, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY) and Herdewijn (2004, Oligonucleotide Synthesis: Methods and Applications (Methods in Molecular Biology), Humana Press, Totowa, New Jersey) It can be prepared (eg, isolated, purified or synthesized) using conventional methods known in the art, such as those described.

  Although the present invention has been described by way of example, it is understood that the terminology used is not limiting and is intended to fall within the scope of the stated language in nature. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as explicitly described herein.

  All disclosures of patents, publications and database registrations cited above are specifically and individually incorporated by reference for such individual patents, publications and registrations, respectively. The entire disclosure is hereby incorporated by reference to the same extent as if individually set forth.

  The following examples illustrate the invention.

  The following construction is performed according to general gene manipulation techniques and molecular cloning techniques detailed in Sambrook et al. (A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY), or using commercially available kits. Follow the manufacturer's recommendations. PCR amplification techniques are known to those skilled in the art (see, eg, PCR protocols -A guide to methods and applications, 1990 published by Innis, Gelfand, Sninsky and White, Academic Press). Recombinant plasmids with ampicillin resistance gene were transformed into E. coli C600 (Stratagene), BJ5183 (Hanahan, 1983, J. Mol. Biol. 166, 557-580) in agar or liquid medium supplemented with 100 μg / ml antibiotic. Duplicate within NM522. The construction of recombinant vaccinia virus is described in the literature cited above, as well as in Macckett et al. (1982, Proc. Natl. Acad. Sci. USA 79, 7415-7419) and Macckett et al. (1984, J. Virol. 49, 857-864) according to conventional methods in this technical field. In order to facilitate the selection of this recombinant vaccinia virus, the E. coli selection gene gpt (xanthine guanine phosphoribosyltransferase) (Falkner and Moss, 1988, J. Virol. 62, 1849-1854) is used.

Example 1 Identification and Evaluation of Novel Signaling Sequences The presentation of antibodies expressed by recombinant MVA was shown to be improved by redirecting the recombinant protein to the plasma membrane surface. For presentation on the plasma membrane, it is necessary that the gene of interest is cloned between a sequence encoding a signal peptide and a sequence encoding a membrane-fixing peptide. Such signaling peptides are generally present in membrane-bound proteins such as rabies glycoproteins of various virus strains and are widely used to express the polypeptide of interest on the cell surface (see WO 99/03885). More specifically, for example, it is obtained as a deposit number M38452 (ERA strain), ay009097 (PG strain), S72465 (Nishigahara strain) and u117751 (artic fox strain) in a specialized data bank such as Uniprot that can be used in NCBI. The amino acid sequences of four different rabies virus strains that can be produced and the encoded glycoprotein are described. However, the combination of signaling peptides derived from these available rabies glycoproteins is not suitable for multigene expression vectors when more than two recombinant polypeptides need to be directed to the plasma membrane surface . Due to their high homology at the nucleotide level, homologous recombination events can occur, adversely affecting the stability of the vector and resulting in vector stocks that are unsuitable for human use.

  The present invention makes it possible to solve this potential homologous recombination problem, a novel signaling peptide derived from rabies glycoprotein, and such engineered to exhibit less than 75% homology with each other. A “degenerated” nucleotide sequence encoding a unique peptide is provided. These sequences were altered by modifying codon usage patterns to reduce DNA homology and thus limit the risk of recombination during the vector production process.

  Six novel nucleotide sequences encoding the signal and membrane-anchoring peptides were engineered, M38452, v1 (SEQ ID NO: 23), ay009097, v1 (SEQ ID NO: 24), S72465, v1 (SEQ ID NO: 25), u117751, v1 ( SEQ ID NO: 26), u11751, v2 (SEQ ID NO: 27) and M38452, v2-ay009097v2 (SEQ ID NO: 28). Table 1 shows the percentage homology shown by the nucleotide sequence of the present invention encoding a signal peptide, and Table 2 shows the percentage homology shown by the nucleotide sequence of the present invention encoding a membrane anchored peptide.

a) Generation of nucleotide sequences encoding these novel signaling sequences These nucleotide sequences were generated by assembling overlapping oligonucleotides. More specifically, in each case, two oligonucleotides were used to generate the sequence encoding the signal peptide and four oligonucleotides were used for the membrane anchoring sequence. Thereafter, each synthetic sequence pair was inserted into a PBS-derived vector, and pTG15833 (S72465, v1), pTG15834 (u117751, v1), pTG15835 (M38452, v1), pTG15943 (ay009097, v1), pTG15944 (M38452, v2-ay009097, v2) and pTG15945 (u117751, v2) were obtained.

b) Evaluation of novel signaling sequences Non-oncogenic HPV-16 E7 protein (deletion of amino acid residues 21-26 (ΔDLYCYE), described in WO 99/03885) is used as a model protein and is encoded by each of six sequence pairs The ability of the signal peptide and membrane-immobilized peptide expressed to express a predetermined polypeptide immobilized on the plasma membrane was evaluated. The nucleotide sequence encoding the HPV-16 E7 variant was cloned into the BamHI site of each plasmid above between the signal peptide coding sequence and the membrane-anchored peptide sequence. In each case, expression was placed under the control of the vaccinia p7.5K promoter (Cochran et al, 1985. J. Virol. 54, 30-37). Next, E7 expression and localization was assessed following transient infection of primary chicken embryo fibroblasts (CEF). 10 ⁶ cells previously infected with MVATGN 33.1 with MOI 1 pfu / cell were transfected with 1 μg of plasmid DNA using Lipofectin (Invitrogen). 24 hours after transfection, the expression level of E7 was assessed by Western blot analysis.

  As a result, E7 was expressed at an equivalent level from each test plasmid except for pTG15833 (sequence S72465v1 corresponding to SEQ ID NO: 25), which shows a lower expression level. This expression level was on the same order as that seen with the control plasmid expressing E7 under the control of the signal obtained from the glycoprotein of the reference rabies strain ERA (M38452) and a membrane-anchored peptide.

Cell localization was assessed by immunofluorescence. 7.5 10 ⁴ BHK21 cells were infected with MVATG33.1 with MOI 1 pfu / cell and then transfected with 0.125 μg of plasmid DNA using Lipofectin (Invitrogen). After 24 hours, immunofluorescence was performed using rabbit polyclonal anti-E7 antibody. E7 protein was detected on the plasma membrane surface for all signaling sequences as expected.

  In conclusion, these novel signaling sequences could be used to express one or more polypeptides of interest immobilized on the plasma membrane on the surface of the host cell.

Table 1: Percentage of DNA homology between sequences encoding the signal peptides disclosed herein.

Table 2: Percentage of DNA homology between sequences encoding membrane-anchored peptides disclosed herein

Claims (35)

  A peptide selected from the group consisting of peptides consisting essentially of or consisting of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.   A polypeptide of interest and (i) a peptide consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or (ii) shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. A peptide consisting essentially of or consisting of an amino acid sequence, or (iii) a peptide consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and SEQ ID NO: 3, SEQ ID NO: A chimeric polypeptide comprising both a peptide consisting essentially of or consisting of the amino acid sequence shown in 4 or SEQ ID NO: 5.   The chimeric polypeptide according to claim 2, wherein the peptide consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is linked at the amino terminus of the chimeric polypeptide.   4. A chimeric polypeptide according to claim 2 (i) or 3, which does not contain other signaling peptides.   The chimeric polypeptide according to claim 2 or 3, comprising at least one membrane-immobilizing peptide.   The chimeric polypeptide according to claim 5, wherein the membrane-fixing peptide is linked at the carboxy terminus of the polypeptide of interest or within the C-terminal portion.   The chimeric polypeptide according to any one of claims 2, 3, 5, and 6, wherein the signal peptide consists of an amino acid sequence represented by SEQ ID NO: 2, and the membrane-immobilized peptide consists of an amino acid sequence represented by SEQ ID NO: 3. peptide.   The chimeric polypeptide according to any one of claims 2, 3, 5, and 6, wherein the signal peptide consists of an amino acid sequence represented by SEQ ID NO: 1, and the membrane-immobilized peptide consists of an amino acid sequence represented by SEQ ID NO: 5. peptide.   The chimeric polypeptide according to any one of claims 2 to 8, wherein the target polypeptide is heterologous to the rabies virus.   The chimeric polypeptide according to claim 9, wherein the polypeptide of interest is selected from the group consisting of an immunogenic polypeptide and an anti-tumor polypeptide.   11. The chimeric polypeptide according to claim 10, wherein the immunogenic polypeptide is a papilloma virus polypeptide.   An isolated nucleic acid molecule comprising at least the nucleotide sequence encoding the peptide of claim 1 or the chimeric polypeptide of any one of claims 2-11.   An isolated nucleic acid molecule comprising at least one of the nucleotide sequences set forth in any of SEQ ID NOs: 11-28.   14. The nucleic acid molecule according to claim 13, wherein the nucleotide sequence encoding the polypeptide of interest is inserted between about number 70 to about number 76 of SEQ ID NOs: 23-28.   15. A nucleic acid molecule according to any one of claims 12 to 14, which is placed under the control of suitable regulatory elements to ensure its expression in a given host cell or organism.   A vector for expressing one or more chimeric polypeptides according to any one of claims 2-11.   17. A vector according to claim 16, comprising one or more nucleic acid molecules according to any one of claims 12-15.   18. A vector according to claim 17, comprising two, three or four nucleic acid molecules according to any one of claims 12-15.   19. A vector according to claim 18, wherein said 2, 3 or 4 nucleic acid molecules exhibit a percentage homology of less than 75% between each other.   The nucleotide sequence of SEQ ID NO: 24 for allowing expression of the first polypeptide of interest on the cell membrane and the nucleotide sequence of SEQ ID NO: 28 for allowing expression of the second polypeptide of interest on the cell membrane 20. A vector according to claim 19 comprising:   The vector according to any one of claims 16 to 20, which is a viral vector or a non-viral vector.   The vector according to claim 21, which is an adenovirus vector.   23. The vector of claim 22, wherein the adenoviral vector is replication defective.   The vector according to claim 21, which is a poxvirus vector.   25. The vector of claim 24, wherein the poxvirus vector is obtained from a vaccinia virus selected from the group consisting of Copenhagen strain, Weiss strain, NYVAC and highly attenuated modified Ankara (MVA) strain.   An infectious virus particle comprising the nucleic acid molecule according to any one of claims 12 to 15 or the vector according to any one of claims 16 to 25.   A host cell comprising the nucleic acid molecule according to any one of claims 12 to 15, the vector according to any one of claims 16 to 25 or the infectious viral particle according to claim 26.   A chimeric polypeptide according to any one of claims 2 to 11, a nucleic acid molecule according to any one of claims 12 to 15, or any one of claims 16 to 25 in a therapeutically effective amount. A pharmaceutical composition comprising the vector of claim 26, the infectious viral particle of claim 26, or the host cell of claim 27, or any combination thereof and a pharmaceutically acceptable vehicle. object.   A chimeric polypeptide according to any one of claims 2 to 11, a nucleic acid molecule according to any one of claims 12 to 15 for the manufacture of a medicament intended for the treatment or prevention of a disease or condition. Or use of a vector according to any one of claims 16 to 25, or an infectious viral particle according to claim 26, or a host cell according to claim 27, or any combination thereof.   30. Use according to claim 29, wherein the disease or condition is an infectious disease.   Use of a peptide consisting essentially of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 as a signal peptide.   Use of a peptide consisting essentially of or consisting of the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 as a membrane-fixing peptide.   A method for recombinant production of a polypeptide comprising the step of: a) introducing a vector according to any one of claims 16 to 25 or an infectious viral particle according to claim 26 into a host cell, b) Culturing the transformed host cell under conditions allowing expression of the polypeptide of interest, and c) recovering the polypeptide from the culture and optionally purifying it.   A method for expressing a polypeptide of interest outside a cell, wherein (a) a first selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 A step of producing a nucleic acid molecule by linking a nucleotide sequence and a second nucleotide sequence encoding the polypeptide (the nucleic acid molecule comprises a signal peptide encoded by the first nucleotide sequence and a target polypeptide at the N-terminus. Encoding a chimeric polypeptide comprising at the C-terminus); (b) inserting the resulting nucleic acid molecule into an expression vector; and (c) introducing the expression vector into a host cell or organism. A method that consists of   A method for expressing a polypeptide immobilized on the surface of a plasma membrane, comprising: (a) selecting from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28 A second nucleotide sequence encoding the polypeptide of interest is inserted into the first nucleotide sequence at any position from the nucleotide at position 70 to the nucleotide at position 76 of the first nucleotide sequence. A step of producing a nucleic acid molecule (the nucleic acid molecule is encoded by a signal peptide encoded by the first nucleotide sequence, a target polypeptide encoded by the second nucleic acid, and the first nucleotide sequence). Obtained from the N-terminus to the C-terminus); (b) obtained Step inserting the acid molecule into an expression vector, and (c) said expression vector comprising the step of introducing into a host cell or organism, methods.